The long term objective of this proposal is to understand the cellular mechanisms of sustained secretion in neuroendocrine cells. Using a combination of new imaging, electrophysiological, and molecular biological techniques, different aspects of the secretion cycle in pancreatic beta-cells will be examined. In particular, single secretory granules and single secretory endosomes will be separately identified and tracked in living cells under a variety of conditions. Specifically, the aims are to test the following hypotheses: (1) Trafficking of secretory granules and secretory-derived endosomes is regulated by cytoplasmic Ca2+ and protein phosphorylation-signals which themselves are generated by cell stimulation from neuronal and hormonal inputs. (2) The readily releasable pool of secretory granules is replenished from predocked granules, without the need for mobilization of granules from the cytoplasm. Sustained secretion, however, requires movement of granules from deep in the cell interior to the plasma membrane. (3) Secretory granules are directly retrieved during endocytosis, and can be recycled to the releasable pool for subsequent exocytosis. Single secretory granules will be identified because they are labeled with a granule specific green fluorescent protein chimera. Single endosomes derived from secretory granules that undergo exo-endocytosis will be additionally labeled with styryl dyes FM1-43 and FM4-64. These optical assays of membrane trafficking in living cells will be combined with patch clamp recording of membrane capacitance and electron microscopy to establish a firm physiological understanding of the mechanisms that allow for sustained secretion from neuroendocrine cells. These studies will lay the ground work for an understanding of how these processes may be altered in diseases of neuroendocrine cells such as diabetes mellitus that demonstrate an apparent disregulation of secretory granule biogenesis, trafficking or recycling.